Labeled Probe and Methods of Use

ABSTRACT

The present disclosure provides for methods and compositions useful for imaging inflammation and inflammatory disease markers with an affinity for TREM-1 antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication Ser. No. 62/254,396, having the title “LABELED PROBE ANDMETHODS OF USE,” filed on Nov. 12, 2015, the disclosure of which isincorporated herein in by reference in its entirety.

BACKGROUND

Molecular imaging using positron emission tomography (PET) has enormouspotential for enhancing our understanding of the in vivo role ofneuroinflammation, and for enabling longitudinal monitoring of bothdisease progression and real-time response to novel therapies thatdirectly or indirectly affect immune processes. Neuroinflammation is akey pathological feature of many central nervous system (CNS) diseases.In human neurological disease, our understanding of the temporaldynamics, the anatomical distribution, and the beneficial versus toxicnature of neuroinflammation is currently very limited. Considering thatneuroinflammation has become a promising target for developingdisease-modifying therapeutics for many of the above listed neurologicalconditions, our ability to quantify and track the neuroinflammatorycomponent of disease via biomarkers is of great importance. Triggeringreceptor expressed on myeloid cells (TREM-1) is an inflammatory membranereceptor that is expressed only on myeloid lineage cells and is uniquein its function as a potent amplifier of toxic inflammatory responses.TREM-1 aggravates the inflammatory response by synergizing with patternrecognition receptors, such as toll-like receptors (TLRs) and nod-likereceptors (NLRs) to amplify pro-inflammatory cytokine production,protease production, and formation of reactive oxygen species (ROS).

Currently the selection of available PET agents for imagingneuroinflammation is limited. Thus, there is a need to developappropriate imaging agents.

SUMMARY

The present disclosure provides for methods and compositions useful forimaging inflammation and inflammatory disease markers with an affinityfor TREM-1 antibodies.

An embodiment of the present disclosure includes a method of imaging aninflammatory disease in a subject, among others, that includes:administering to a subject a labeled probe in a detectably effectiveamount, wherein the labeled probe includes an agent having an affinityfor TREM-1 and a radiolabel, imaging at least a portion of the subject;and detecting the labeled probe, wherein the location of the labeledprobe corresponds to inflammation corresponding to the inflammatorydisease. In an embodiment, the inflammatory disease is selected from thegroup consisting of: Alzheimers disease, multiple sclerosis, epilepsy,traumatic brain injury, cancer, arthritis, inflammatory bowel disease,Huntington's disease, ALS, Parkinson's disease, an infectious disease,sepsis, pain, stroke, chronic fatigue syndrome, depression,schizophrenia, and a CNS or peripheral inflammatory disease. In anembodiment, the radiolabel can include: ⁶⁴Cu, ¹²⁴I, ^(76/77)Br, ⁸⁶Y,⁸⁹Zr, ⁶⁸Ga, ¹⁸F, ¹¹C, ¹²⁵I, ¹²⁴I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁶⁸Ga,⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ⁸⁶Y, ¹⁷⁷Lu, and¹⁵³Sm. In an embodiment, the radiolabel is conjugated to the agonistantibody of TREM-1 using a chelator such as:(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); NOTA(1,4,7-Triazacyclononane-1,4,7-triacetic acid); EDTA(Ethylenediaminetetraacetic acid); Df (Desferrioxamine); DTPA(Diethylenetriaminepentaacetic acid; TETA (Triethylenetetramine).

An embodiment of the present disclosure can include a method fordiagnosing the presence of an inflammatory disease that includes:administering to a subject a labeled probe in a detectably effectiveamount, wherein the labeled probe includes an agent having an affinityfor TREM-1 and a radiolabel; imaging at least a portion of the subject;and detecting the labeled probe, wherein the location of the labeledprobe corresponds to inflammation corresponding to the inflammatorydisease, wherein detection of the labeled probe in a location above athreshold is an indication of presence of the inflammatory disease atthe location.

An embodiment of the present disclosure can include a method ofmonitoring the progress of an inflammatory disease in a subject thatincludes: administering to a subject a labeled probe, wherein thelabeled probe includes an agent having an affinity for TREM-1 and aradiolabel; imaging at least a portion of the subject; and detecting thelabeled probe, wherein the location of the labeled probe corresponds toinflammation corresponding to the inflammatory disease, wherein thedimensions of the location are monitored over time. In an embodiment,the level of uptake of the labeled probe in a tissue corresponds to thelevel inflammation in the tissue.

An embodiment of the present disclosure includes a labeled probeincluding an agent having an affinity for TREM-1 and a radiolabel.

An embodiment of the present disclosure includes a pharmaceuticalcomposition, including: a pharmaceutical carrier and an effective doseof a labeled probe, wherein the labeled probe includes an agent havingan affinity for TREM-1 and a radiolabel.

Other compositions, methods, features, and advantages will be or becomeapparent to one with skill in the art upon examination of the followingdrawings and detailed description. It is intended that all suchadditional compositions, methods, features and advantages be includedwithin this description, be within the scope of the present disclosure,and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readilyappreciated upon review of the detailed description of its variousembodiments, described below, when taken in conjunction with theaccompanying drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure.

FIG. 1 illustrates the structure of the probe and radiochemistryresults.

FIGS. 2A-H are fluorescent images of TREM-1 transfection using HEKcells.

FIG. 3 shows the results of [⁶⁴Cu]TREM-1 cell binding studies.

FIG. 4 compares imaging from PET and CT modalities of wild-type mice at3 hours post-injection of [⁶⁴Cu]TREM-1.

FIG. 5 shows PET and CT images demonstrating visualization of TREM-1 inMCAo stroke vs. sham mice at 19 hours post-injection of [⁶⁴Cu]TREM-1.

FIG. 6 shows the quantitation of [⁶⁴Cu]TREM-1 PET signal in brainregions from each hemisphere of MCAo stroke versus sham mice at 19 hourspost-injection.

FIG. 7 shows ex vivo biodistribution of [⁶⁴Cu]TREM-1 in organs from MCAoand sham mice 19 hours post-injection of tracer (and 2 days after strokesurgery).

FIGS. 8A-B demonstrate [⁶⁴Cu]TREM1-mAb PET tracer binds to TREM1 invitro with high specificity. FIG. 8A is fluorescent microscopy of TREM1in HEK293 cells+/−TREM1 transfection. FIG. 8B shows percent binding oftracer (normalized to μg protein) in TREM1 transfected versusuntransfected cells.

FIG. 9 is in vivo PET imaging of LPS-induced sepsis using[⁶⁴Cu]TREM1-mAb. Images were acquired 24 h after i.p. injection of LPSor vehicle, and are displayed as a maximum intensity projection.

FIG. 10 demonstrates significantly higher uptake of [⁶⁴Cu]TREM1-mAb inspleen and liver of mice 24 h post injection of LPS compared to vehicletreated mice and LPS mice injected with radiolabeled isotope control.Results are from in vivo PET quantification 24 h after mice receivedi.p. injection of LPS (5 mg/kg) or saline alone.

FIG. 11 demonstrates significantly higher uptake of [⁶⁴Cu]TREM1-mAb inbrain, lung, liver, and spleen of mice with LPS-induced sepsis. Resultsare from ex vivo gamma counting of tissues 24 h after mice received i.p.injection of LPS (5 mg/kg) or vehicle, n=6-8 per group.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, synthetic organic chemistry,biochemistry, biology, molecular biology, molecular imaging, and thelike, which are within the skill of the art.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the probes disclosed and claimed herein.Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C., and pressure is at or near atmospheric. Standardtemperature and pressure are defined as 20° C. and 1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Definitions

In describing and claiming the disclosed subject matter, the followingterminology will be used in accordance with the definitions set forthbelow.

As used herein, “antibody” refers to a protein produced by B cells thatis used by the immune system to identify and neutralize foreigncompounds, which are also known as antigens. Antibodies areglycoproteins belonging to the immunoglobulin superfamily. Antibodies,recognize and bind to specific epitopes on an antigen.

By “administration” or “administering” is meant introducing a probe or alabeled probe of the present disclosure into a subject. The preferredroute of administration of the compounds is intravenous. However, anyroute of administration, such as oral, topical, subcutaneous,peritoneal, intraarterial, inhalation, vaginal, rectal, nasal,introduction into the cerebrospinal fluid (e.g., intrathecal), orinstillation into body compartments can be used.

In accordance with the present disclosure, “a detectably effectiveamount” of the labeled probe of the present disclosure is defined as anamount sufficient to yield an acceptable image using equipment that isavailable for clinical use. A detectably effective amount of the labeledprobe of the present disclosure may be administered in more than oneinjection. The detectably effective amount of the labeled probe of thepresent disclosure can vary according to factors such as the degree ofsusceptibility of the individual, the age, sex, and weight of theindividual, idiosyncratic responses of the individual, and the like.Detectably effective amounts of the probe of the present disclosure canalso vary according to instrument and film-related factors. Optimizationof such factors is well within the level of skill in the art.

As used herein, the term “subject” includes vertebrates such as humansand mammals (e.g., cats, dogs, horses, etc.). Typical subjects to whichembodiments of the present disclosure may be administered will bemammals, particularly primates, especially humans. For veterinaryapplications, a wide variety of subjects will be suitable, e.g.,livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids and cell samples of the abovesubjects will be suitable for use, such as mammalian (particularlyprimate such as human) blood, urine, or tissue samples, or blood, urine,or tissue samples of the animals mentioned for veterinary applications.In some embodiments, a system includes a sample and a subject. The term“living subject” refers to a subject noted above that is alive and isnot dead. The term “living subject” refers to the entire subject and notjust a part excised (e.g., a liver or other organ) from the livingsubject.

The term “detectable” refers to the ability to detect a signal over thebackground signal.

The term “detectable signal” is a signal derived from non-invasiveimaging techniques such as, but not limited to, positron emissiontomography (PET). The detectable signal is detectable anddistinguishable from other background signals that may be generated fromthe subject. In other words, there is a measurable and statisticallysignificant difference (e.g., a statistically significant difference isenough of a difference to distinguish among the detectable signal andthe background, such as about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%,or 40% or more difference between the detectable signal and thebackground) between the detectable signal and the background. Standardsand/or calibration curves can be used to determine the relativeintensity of the detectable signal and/or the background.

The term “pharmaceutically acceptable carrier” as used herein refers toa diluent, adjuvant, excipient, or vehicle with which a probe of thedisclosure is administered and which is approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. The pharmaceutical carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. When administered to a patient, the probes of thedisclosure and pharmaceutically acceptable carriers preferably should besterile. Water is a useful carrier when the probe of the disclosure isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical carriers also includeexcipients such as glucose, lactose, sucrose, glycerol monostearate,sodium chloride, glycerol, propylene, glycol, water, ethanol and thelike. The present compositions, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thepresent compositions advantageously may take the form of solutions,emulsion, sustained-release formulations, or any other form suitable foruse.

The disclosure encompasses compositions and dosage forms of thecompositions of the disclosure that can include one or more compoundsthat reduce the rate by which an active ingredient will decompose. Suchcompounds, which are referred to herein as “stabilizers,” include, butare not limited to, antioxidants such as ascorbic acid, pH buffers, orsalt buffers. In addition, pharmaceutical compositions or dosage formsof the disclosure may contain one or more solubility modulators, such assodium chloride, sodium sulfate, sodium or potassium phosphate, ororganic acids. An exemplary solubility modulator is tartaric acid.

“Pharmaceutically acceptable salt” refers to those salts that retain thebiological effectiveness and properties of the free bases and that areobtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,succinic acid, tartaric acid, citric acid, and the like.

Embodiments of the present disclosure include pharmaceuticalcompositions that include the labeled probe, pharmaceutically acceptablesalts thereof, with other chemical components, such as physiologicallyacceptable carriers and excipients. One purpose of a pharmaceuticalcomposition is to facilitate administration of labeled probe to asubject (e.g., human).

Embodiments of the present disclosure may be salts and these salts arewithin the scope of the present disclosure. Reference to a compound ofany of the formulas herein is understood to include reference to saltsthereof, unless otherwise indicated. The term “salt(s)”, as employedherein, denotes acidic and/or basic salts formed with inorganic and/ororganic acids and bases. In addition, when an embodiment of the presentdisclosure contains both a basic moiety and an acidic moiety,zwitterions (“inner salts”) may be formed and are included within theterm “salt(s)” as used herein. Pharmaceutically acceptable (e.g.,non-toxic, physiologically acceptable) salts are preferred, althoughother salts are also useful, e.g., in isolation or purification stepswhich may be employed during preparation. Salts of the compounds may beformed, for example, by reacting an active compound with an amount ofacid or base, such as an equivalent amount, in a medium such as one inwhich the salt precipitates or in an aqueous medium followed bylyophilization.

Embodiments of the present disclosure that contain a basic moiety mayform salts with a variety of organic and inorganic acids. Exemplary acidaddition salts include acetates (such as those formed with acetic acidor trihaloacetic acid, for example, trifluoroacetic acid), adipates,alginates, ascorbates, aspartates, benzoates, benzenesulfonates,bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides (formed withhydrochloric acid), hydrobromides (formed with hydrogen bromide),hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed withmaleic acid), methanesulfonates (formed with methanesulfonic acid),2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates,persulfates, 3-phenylpropionates, phosphates, picrates, pivalates,propionates, salicylates, succinates, sulfates (such as those formedwith sulfuric acid), sulfonates (such as those mentioned herein),tartrates, thiocyanates, toluenesulfonates such as tosylates,undecanoates, and the like.

Embodiments of the present disclosure that contain an acidic moiety mayform salts with a variety of organic and inorganic bases. Exemplarybasic salts include ammonium salts, alkali metal salts such as sodium,lithium, and potassium salts, alkaline earth metal salts such as calciumand magnesium salts, salts with organic bases (for example, organicamines) such as benzathines, dicyclohexylamines, hydrabamines (formedwith N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine, and the like.

Basic nitrogen-containing groups may be quaternized with agents such aslower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides,bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl,dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides), aralkyl halides(e.g., benzyl and phenethyl bromides), and others.

Solvates of the compounds of the disclosure are also contemplatedherein. Solvates of the compounds are preferably hydrates.

The amounts and a specific type of active ingredient (e.g., a labeledprobe) in a dosage form may differ depending on various factors. It willbe understood, however, that the total daily usage of the compositionsof the present disclosure will be decided by the attending physician orother attending professional within the scope of sound medical judgment.The specific effective dose level for any particular subject will dependupon a variety of factors, including for example, the activity of thespecific composition employed; the specific composition employed; theage, body weight, general health, sex, and diet of the subject; the timeof administration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; theexistence of other drugs used in combination or coincidental with thespecific composition employed; and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the composition at levels lower than those required toachieve the desired effect and to gradually increase the dosage untilthe desired effect is achieved.

The term “positron emission tomography” as used herein refers to anuclear medicine imaging technique that produces a three-dimensionalimage or map of functional/molecular processes in the body. The systemdetects pairs of gamma rays emitted indirectly by a positron-emittingradioisotope, which is introduced into the body via a molecule specificfor a target/process of interest. Images of the target/process ofinterest in space are then reconstructed by computer analysis. Usingstatistics collected from tens-of-thousands of coincidence events, a setof simultaneous equations for the total activity of each parcel oftissue can be solved by a number of techniques, and a map ofradioactivities as a function of location for parcels or bits of tissuemay be constructed and plotted. The resulting map shows the tissues inwhich the molecular probe has become concentrated. PET technology can beused to trace the biologic pathway of any compound in living humans (andmany other species as well), provided it can be radiolabeled with a PETisotope. The half-life of fluorine-18, copper-64, and zirconium-89 arelong enough such that imaging agents labeled with these radioisotopescan be manufactured commercially at an offsite location. Otherradioisotopes may have shorter half-lives, such as carbon-11 (about 20min), nitrogen-13 (about 10 min), oxygen-15 (about 2 min), andfluorine-18 (about 110 min).

The term “label” as used herein refers to any moiety that may be linked(e.g. bonded or otherwise associated with) to the agent (e.g., antibodyof TREM-1) of the present disclosure and which may be used to provide adetectable image including, but not limited to, a radiolabel such as aPET probe.

The term “in vivo imaging” as used herein refers to methods or processesin which the structural, functional, molecular, or physiological stateof a living being is examinable without the need for a life-endingsacrifice.

The term “non-invasive in vivo imaging” as used herein refers to methodsor processes in which the structural, functional, molecular, orphysiological state of being is examinable by remote physical probingwithout the need for breaching the physical integrity of the outer(skin) or inner (accessible orifices) surfaces of the body.

Abbreviations

Abbreviations used herein may include: DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); NOTA(1,4,7-Triazacyclononane-1,4,7-triacetic acid); EDTA(Ethylenediaminetetraacetic acid); Df (Desferrioxamine); DTPA(Diethylenetriaminepentaacetic acid; TETA (Triethylenetetramine).

General Discussion

Embodiments of the present disclosure provide for labeled probes (e.g.,a ⁶⁴Cu-DOTA TREM-1 probe), methods of making labeled probes,pharmaceutical compositions including labeled probes, methods of usinglabeled probes; methods of diagnosing, localizing, monitoring, and/orassessing inflammatory diseases or related biological events usinglabeled probes; kits for diagnosing, localizing, monitoring, and/orassessing inflammatory diseases or related biological events, and thelike. In particular, the present disclosure includes methods relating tonon-invasive imaging (e.g., using a positron emission tomography (PET)imaging system) using labeled probes (e.g., a ⁶⁴Cu-DOTA TREM-1 probe),in vivo. Additional details are described herein and in the Examples.

Embodiments of the present disclosure provide for the first labeledprobe targeting TREM1 for neuroinflammation imaging. Most PET agentscurrently used to image neuroinflammation were not originallysynthesized for that purpose. For example, TSPO tracers were firstsynthesized in 1984 to investigate the role of the peripheralbenzodiazepine receptor in cardiovascular, renal, and cerebralpathologies. The present disclosure used an approach that is uniquesince it used unbiased transcriptome analyses from studies ofneurological disease to identify a promising PET imaging biomarker ofneuroinflammation. Since TREM1 is strongly linked to maladaptivemicroglial responses (generation of pro-inflammatory cytokines and freeradicals), as opposed to beneficial functions (e.g., phagocytosis,trophic factor support), this target can serve as an in vivo biomarkerof microglial/macrophage functional status. None of the currentlyavailable neuroinflammation PET tracers can provide this type ofinformation. In addition, embodiments of the present disclosure providefor a labeled probe that is a neuroinflammatory target that isselectively expressed on myeloid cells.

Embodiments of the present disclosure are advantageous for at least thefollowing reasons. In an embodiment, the labeled probe specifically andselectively binds to TREM-1 in vivo and in vitro. In an embodiment, thesample can be imaged via PET imaging. Embodiments of the presentdisclosure provide for non-invasive, early detection, staging, andmonitoring of inflammatory diseases including but not limited toAlzheimer's disease (AD), sepsis, sepsis induced encephalopathy (SIE),multiple sclerosis, epilepsy, traumatic brain injury, cancer, arthritis,inflammatory bowel diseases (e.g., colitis), Huntington's disease,Amytrophic Lateral Sclerosis (ALS), Parkinson's disease, infectiousdiseases, pain, stroke, chronic fatigue syndrome, depression,schizophrenia, and a range of other central nervous system andperipheral inflammatory diseases.

Embodiments of the present disclosure include methods for imaging asample (e.g., tissue or cell(s)) or a subject, that includes contactinga sample with or administering to a subject a labeled probe (e.g.,⁶⁴Cu-DOTA TREM-1) and imaging the sample with a PET imaging system.Imaging can be performed in vivo, ex vivo, and/or in vitro. Inparticular, embodiments of the present disclosure can be used to imageinflammatory diseases or related biological events. In this regard, thesample or subject can be tested to determine if the sample or subjectincludes a inflammatory diseases or related biological conditions, tomonitor the progression (or regression) of the inflammatory diseases, orto assess the response of the inflammatory diseases to treatment, toimage, and the like. In an embodiment, the tissue or cells can be withina subject or can have been removed from a subject.

In an embodiment, the labeled probe (⁶⁴Cu-DOTA TREM-1) can be imagedusing imaging systems such as a positron emission tomography (PET)imaging systems (and combined PET/CT and PET/MR systems) or an exvivo/in vitro phosphor imager. In an embodiment, PET imaging is apreferred embodiment. Other types of labeled probes can use appropriateimaging systems.

In an embodiment, the labeled probe can be used in imaging, diagnosing,localizing, monitoring, and/or assessing inflammatory diseases andrelated biological events as well as tracking response to therapy. Forexample, embodiments of the present disclosure can be used for imaging,diagnosing, localizing, monitoring, and/or assessing: Alzheimer'sdisease (AD), multiple sclerosis, epilepsy, traumatic brain injury,cancer, arthritis, inflammatory bowel disease, Huntington's disease,ALS, Parkinson's disease, an infectious disease, sepsis, pain, stroke,chronic fatigue syndrome, depression, schizophrenia, and a CNS orperipheral inflammatory disease. In an embodiment the inflammatorydisease can be Alzheimer's disease. In particular, the presentdisclosure includes methods relating to non-invasive imaging (e.g.,using positron emission tomography (PET) imaging system) using thelabeled probe in vivo.

TREM-1 is a biomarker of toxic or maladaptive inflammation. TREM-1 is ahighly regulated gene tightly associated with microglial maladaptiveresponses. TREM-1 is expressed in myeloid lineage cells and is a potentamplifier of pro-inflammatory responses. TREM-1 signals through adapterprotein DAP12/TYROBP, a critical signaling node that emerged in ADgene-regulatory network analyses. Not only does TREM-1 function in thepathogenesis of AD, but it may also serve as a unique inflammatorymarker for maladaptive microglia. Under physiologic conditions, myeloidcells express low levels of TREM-1; however, during pro-inflammatoryresponses, TREM-1 expression is selectively upregulated in microglia.

The present disclosure describes non-invasive imaging of TREM-1 and itsinvolvement in toxic/maladaptive inflammation in living subjects. In aparticular embodiment, a radiolabeled agent (e.g., agonist antibody) forTREM-1 with a PET radioisotope (e.g., copper-64) is provided that canspecifically and selectively bind to TREM-1 in vitro in cellstransfected with TREM-1 and in vivo in a living subject.

Embodiments of the present disclosure include a labeled probe in achemical composition, a pharmaceutical composition, or the like. In anembodiment, the labeled probe includes an agent having an affinity forTREM-1 and a radiolabel. In an embodiment, the agent can have anaffinity (e.g., a preferential attraction or specific attraction theretosubstantially to the exclusion of other proteins and the like associatedwith inflammatory disease) for TREM-1. In an embodiment, the agent canbe a TREM-1 antibody (e.g., humanized or mouse TREM-1 antibody such asone that can be purchased from R&D Systems (e.g., Mouse TREM-1 Antibody,Catalog Number: MAB1187, Human TREM-1 Antibody, Catalog Number:MAB1278), Hycult Biotech, and the like), an engineered fragment of theTREM-1 antibody, a TREM-1 peptide, or a small molecule associated withTREM-1. In an embodiment, the TREM-1 antibody can be a TREM-1 agonistantibody or an antagonist TREM-1 antibody. These monoclonal antibodies(mAbs) have been shown to link directly to the extracellular domain ofTREM-1.

In an embodiment, the labeled probe includes on or more radiolabels. Inan exemplary embodiment, the radiolabel can include one or more of thefollowing: ⁶⁴Cu, ¹²⁴I, ^(76/77)Br, ⁸⁶Y, ⁸⁹Zr, ⁶⁸Ga, ¹⁸F, ¹¹C, ¹²⁵I,¹²⁴I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁶⁸Ga, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br,⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ⁸⁶Y, ¹⁷⁷Lu, ¹⁵³Sm, ¹⁴C, or ³H. In anembodiment, the radiolabel can be ⁶⁴Cu, ¹²⁴I, ^(76/77)Br, ⁸⁶Y, ⁸⁹Zr,¹⁸F, or ⁶⁸Ga. In an embodiment, the radiolabel can be ⁶⁴Cu.

In an embodiment, the radiolabel can be chelated with the agent using achelator. In an embodiment, the chelator can have a direct bond to anamino acid or indirect bond (e.g., using a linker) to the agent. Forexample, the chelator can form a bond to an amino acid of an antibody ofTREM-1 such as a lysine group in the antibody or other appropriate groupto form a bond. One of skill in the art can select the appropriate bondor linker to be used in a particular situation to retain the propertiesof the agent, the radiolabel, and the TREM-1 imaging probe. In anembodiment, 1, 2, 3, 4, or 5 radiolabels can be present in the labeledprobe. In an embodiment, the radiolabels can be chelated to the sequenceusing a chelator such as 1, 4, 7, 10-tetraazadodecane-N, N′; N″,N″-tetraacetic acid (DOTA); 1, 4, 7-triazacyclononane-1, 4, 7-triaceticacid (NOTA); 1,4,8,11-tetraazacyclotetradecane-1, 4, 8, 11-tetraaceticacid (TETA); diethylenetriaminepentaacetic (DTPA),ethylenediaminetetraacetic acid (EDTA), and desferrioxamine (Df),1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), and derivativesof each of these. In an embodiment, the chelator can be DOTA andoptionally bonded via a lysine.

In an embodiment, the ⁶⁴Cu-DOTA TREM-1 imaging probe includes a label,⁶⁴Cu, that can be used to detect, image, or otherwise identify the⁶⁴Cu-DOTA TREM-1 probe, quantify the amount of ⁶⁴Cu-DOTA TREM-1 probe,determine the location of the ⁶⁴Cu-DOTA TREM-1 probe (e.g., in imaging),and combinations thereof. In an embodiment, the chelator for the⁶⁴Cu-DOTA TREM-1 probe is DOTA.

Methods of Use

Embodiments of this disclosure include, but are not limited to: methodsof imaging a sample or a subject using the labeled probe (e.g.,⁶⁴Cu-DOTA TREM-1 probe); methods of imaging inflammatory disease orrelated biological conditions using the labeled probe (e.g., ⁶⁴Cu-DOTATREM-1 probe); methods of diagnosing inflammatory disease or relatedbiological conditions using the labeled probe (e.g., ⁶⁴Cu-DOTA TREM-1probe); methods of monitoring the progress of inflammatory disease orrelated biological conditions using the labeled probe (e.g., ⁶⁴Cu-DOTATREM-1 probe), and the like.

Embodiments of the present disclosure can be used to image, detect,study, monitor, evaluate, assess, and/or screen, inflammatory disease(e.g., Alzheimer's disease, multiple sclerosis, epilepsy, traumaticbrain injury, cancer, arthritis, inflammatory bowel disease,Huntington's disease, ALS, Parkinson's disease, an infectious disease,sepsis, pain, stroke, chronic fatigue syndrome, depression,schizophrenia, and a CNS or peripheral inflammatory disease) or relatedbiological conditions in vivo or in vitro using the labeled probe (e.g.,⁶⁴Cu-DOTA TREM-1 probe). For example, when the labeled probe isassociated with TREM-1, a detectable signal is emitted that issignificantly different from the background signal (e.g., non-inflamedareas) above a certain threshold, indicating the presence ofinflammatory disease, where the threshold depends upon various variablessuch as the individual subject, the inflammatory disease, and the like.

In a particular embodiment, the ⁶⁴Cu-DOTA TREM-1 probe can be used inimaging Alzheimer's disease. For example, the ⁶⁴Cu-DOTA TREM-1 probe isprovided or administered to a subject in an amount effective to resultin association of the ⁶⁴Cu-DOTA TREM-1 probe into TREM-1. The subject isthen introduced to an appropriate imaging system (e.g., PET system) fora certain amount of time (e.g., this depends on radioisotope beingused). The ⁶⁴Cu-DOTA TREM-1 probe becomes associated with TREM-1 and isdetected using the imaging system. The location of the detected signalfrom the ⁶⁴Cu-DOTA TREM-1 probe can be correlated with the location ofthe inflammation associated with the Alzheimer's disease. In anembodiment, the dimensions of the location can be determined as well.Other labeled probes of the present disclosure can be used in a similarmanner.

In an embodiment, the steps of this method can be repeated at determinedintervals so that the location and/or size/stage of the disease can bemonitored as a function of time and/or treatment. In particular, the⁶⁴Cu-DOTA TREM-1 probe can find use in a subject undergoing treatment(e.g., using a drug, etc.), to aid in visualizing the response of theAlzheimer's disease to the treatment. In this embodiment, the ⁶⁴Cu-DOTATREM-1 probe is typically visualized and the imaging signal isquantified prior to treatment, and periodically (e.g., daily, weekly,monthly, intervals in between these, and the like) during treatment, andthe like, to monitor the regions and extent of the inflammation. Otherlabeled probes can be used in a similar manner.

In an embodiment, the method can be used to select appropriate patientsto receive therapy. For example, a patient can be given ananti-neuroinflammatory drug, and the inflammation can be measured todetermine if the anti-neuroinflammatory drug is having the desiredresults in the patient.

In an embodiment, the method can be used to design clinical trials ofnew anti-neuroinflammatory drugs by measuring the change in inflammationin the area of inflammation when administered the newanti-neuroinflammatory drug over the desired time frame.

It should be noted that the amount effective to result in uptake orassociation of the labeled probe (e.g., ⁶⁴Cu-DOTA TREM-1 imaging probe)into the cells or tissue of interest may depend upon a variety offactors, including for example, the age, body weight, general health,sex, and diet of the subject; the time of administration; the route ofadministration; the rate of excretion of the specific probe employed;the duration of the treatment; the existence of other drugs used incombination or coincidental with the specific composition employed; andlike factors, well known in the medical arts.

Kits

The present disclosure also provides packaged compositions orpharmaceutical compositions comprising a pharmaceutically acceptablecarrier and a labeled probe (e.g., ⁶⁴Cu-DOTA TREM-1 probe) of thedisclosure. In certain embodiments, the packaged compositions orpharmaceutical composition includes the reaction precursors to be usedto generate the labeled probe according to the present disclosure. Otherpackaged compositions or pharmaceutical compositions provided by thepresent disclosure further include materials including at least one of:instructions for using the labeled probe to image a subject, or subjectsamples (e.g., cells or tissues), which can be used as an indicator ofconditions including, but not limited to, inflammatory diseases andbiological related conditions.

Embodiments of this disclosure encompass kits that include, but are notlimited to, the labeled probe (e.g., ⁶⁴Cu-DOTA TREM-1 imaging probe) anddirections (written instructions for their use). The components listedabove can be tailored to the particular biological condition to bemonitored as described herein. The kit can further include appropriatebuffers and reagents known in the art for administering variouscombinations of the components listed above to the subject cell orsubject organism. The imaging probe and carrier may be provided insolution or in lyophilized form. When the imaging probe and carrier ofthe kit are in lyophilized form, the kit may optionally contain asterile and physiologically acceptable reconstitution medium such aswater, saline, buffered saline, and the like.

Dosage Forms

Embodiments of the present disclosure can be included in one or more ofthe dosage forms mentioned herein. Unit dosage forms of thepharmaceutical compositions (the “composition” includes at least thelabeled probe of the present disclosure, e.g., ⁶⁴Cu-DOTA TREM-1 imagingprobe) of this disclosure may be suitable for oral, mucosal (e.g.,nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,intramuscular, subcutaneous, intravenous, intra-arterial, or bolusinjection), topical, or transdermal administration to a patient.Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as hard gelatin capsules and soft elasticgelatin capsules; cachets; troches; lozenges; dispersions;suppositories; ointments; cataplasms (poultices); pastes; powders;dressings; creams; plasters; solutions; patches; aerosols (e.g., nasalsprays or inhalers); gels; liquid dosage forms suitable for oral ormucosal administration to a patient, including suspensions (e.g.,aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, orwater-in-oil liquid emulsions), solutions, and elixirs; liquid dosageforms suitable for parenteral administration to a patient; and sterilesolids (e.g., crystalline or amorphous solids) that can be reconstitutedto provide liquid dosage forms suitable for parenteral administration toa patient.

The composition, shape, and type of dosage forms of the compositions ofthe disclosure typically vary depending on their use. For example, aparenteral dosage form may contain smaller amounts of the activeingredient than an oral dosage form used to treat the same condition ordisorder. These and other ways in which specific dosage formsencompassed by this disclosure vary from one another will be readilyapparent to those skilled in the art (See, e.g., Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990)).

Typical compositions and dosage forms of the compositions of thedisclosure can include one or more excipients. Suitable excipients arewell known to those skilled in the art of pharmacy or pharmaceutics, andnon-limiting examples of suitable excipients are provided herein.Whether a particular excipient is suitable for incorporation into acomposition or dosage form depends on a variety of factors well known inthe art including, but not limited to, the way in which the dosage formwill be administered to a patient. For example, oral dosage forms, suchas tablets or capsules, may contain excipients not suited for use inparenteral dosage forms. The suitability of a particular excipient mayalso depend on the specific active ingredients in the dosage form. Forexample, the decomposition of some active ingredients can be acceleratedby some excipients, such as lactose, or by exposure to water. Activeingredients that include primary or secondary amines are particularlysusceptible to such accelerated decomposition.

The disclosure encompasses compositions and dosage forms of thecompositions of the disclosure that can include one or more compoundsthat reduce the rate by which an active ingredient will decompose. Suchcompounds, which are referred to herein as “stabilizers,” include, butare not limited to, antioxidants such as ascorbic acid, pH buffers, orsalt buffers. In addition, pharmaceutical compositions or dosage formsof the disclosure may contain one or more solubility modulators, such assodium chloride, sodium sulfate, sodium or potassium phosphate, ororganic acids. An exemplary solubility modulator is tartaric acid.

“Pharmaceutically acceptable salt” refers to those salts that retain thebiological effectiveness and properties of the free bases and that areobtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,succinic acid, tartaric acid, citric acid, and the like.

Embodiments of the present disclosure include pharmaceuticalcompositions that include the labeled probe (e.g., ⁶⁴Cu-DOTA TREM-1probe), pharmaceutically acceptable salts thereof, with other chemicalcomponents, such as physiologically acceptable carriers and excipients.One purpose of a pharmaceutical composition is to facilitateadministration of labeled probe (e.g., ⁶⁴Cu-DOTA TREM-1 imaging probe)to a subject (e.g., human).

Embodiments of the present disclosure may be salts and these salts arewithin the scope of the present disclosure. Reference to a compound ofany of the formulas herein is understood to include reference to saltsthereof, unless otherwise indicated. The term “salt(s)”, as employedherein, denotes acidic and/or basic salts formed with inorganic and/ororganic acids and bases. In addition, when an embodiment of the presentdisclosure contains both a basic moiety and an acidic moiety,zwitterions (“inner salts”) may be formed and are included within theterm “salt(s)” as used herein. Pharmaceutically acceptable (e.g.,non-toxic, physiologically acceptable) salts are preferred, althoughother salts are also useful, e.g., in isolation or purification stepswhich may be employed during preparation. Salts of the compounds of anactive compound may be formed, for example, by reacting an activecompound with an amount of acid or base, such as an equivalent amount,in a medium such as one in which the salt precipitates or in an aqueousmedium followed by lyophilization.

Embodiments of the present disclosure that contain a basic moiety mayform salts with a variety of organic and inorganic acids. Exemplary acidaddition salts include acetates (such as those formed with acetic acidor trihaloacetic acid, for example, trifluoroacetic acid), adipates,alginates, ascorbates, aspartates, benzoates, benzenesulfonates,bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides (formed withhydrochloric acid), hydrobromides (formed with hydrogen bromide),hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed withmaleic acid), methanesulfonates (formed with methanesulfonic acid),2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates,persulfates, 3-phenylpropionates, phosphates, picrates, pivalates,propionates, salicylates, succinates, sulfates (such as those formedwith sulfuric acid), sulfonates (such as those mentioned herein),tartrates, thiocyanates, toluenesulfonates such as tosylates,undecanoates, and the like.

Embodiments of the present disclosure that contain an acidic moiety mayform salts with a variety of organic and inorganic bases. Exemplarybasic salts include ammonium salts, alkali metal salts such as sodium,lithium, and potassium salts, alkaline earth metal salts such as calciumand magnesium salts, salts with organic bases (for example, organicamines) such as benzathines, dicyclohexylamines, hydrabamines (formedwith N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine, and the like.

Basic nitrogen-containing groups may be quaternized with agents such aslower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides,bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl,dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides), aralkyl halides(e.g., benzyl and phenethyl bromides), and others.

Solvates of the compounds of the disclosure are also contemplatedherein. Solvates of the compounds are preferably hydrates.

The amounts and a specific type of active ingredient (e.g., a labeledprobe such as ⁶⁴Cu-DOTA TREM-1 probe) in a dosage form may differdepending on various factors. It will be understood, however, that thetotal daily usage of the compositions of the present disclosure will bedecided by the attending physician or other attending professionalwithin the scope of sound medical judgment. The specific effective doselevel for any particular subject will depend upon a variety of factors,including for example, the activity of the specific compositionemployed; the specific composition employed; the age, body weight,general health, sex, and diet of the subject; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; theexistence of other drugs used in combination or coincidental with thespecific composition employed; and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the composition at levels lower than those required toachieve the desired effect and to gradually increase the dosage untilthe desired effect is achieved.

EXAMPLES Example 1: Radiochemistry

Results of the radiochemistry analyses are shown in FIG. 1.

Methods: DOTA conjugation was performed according to establishedprotocols, using metal-free buffers. After conjugation, matrix-assistedlaser desorption/ionization (MALDI) mass spectrometry was conducted todetermine the average number of DOTA molecules conjugated per TREM-1antibody. Subsequently, the DOTA-conjugated TREM-1 mAb was radiolabeledwith ⁶⁴Cu by incubating it in a [⁶⁴Cu]CuCl₂ solution (pH 5.5) at 37° C.for one hour with continual shaking. The reaction was purified via aNAP5 column and specific activity of the final labeled antibody wasdetermined via size exclusion high-performance liquid chromatography(HPLC).

Results: MALDI results showed there are 1.8 DOTA molecules per TREM-1antibody. [⁶⁴Cu]TREM-1 can be synthesized with high specificradioactivity (>75 GBq/μmol), radiochemical purity (>99%), and labelingefficiency (50-75%), which is sufficient for in vitro and in vivo use.

Example 2: In Vitro Cell Binding

Methods: HEK293 cells were plated in 24-well plates 48 h beforetransfection w/TREM-1. Untransfected HEK293 cells were used as controls.Some cells were blocked w/TREM-1 antibody 30 min prior to incubationwith the [⁶⁴Cu]TREM-1 antibody. All cells were incubated with 5 uCi of[⁶⁴Cu]TREM-1 antibody per well for 1 h or 2 h. Cells were washed withPBS, lysed with RIPA buffer, and the activity counted in a gammacounter. Radioactive counts were normalized to the amount of protein ineach well via BCA assay.

Results: FIG. 2 shows that [⁶⁴Cu]TREM-1 displayed >24-fold higherbinding in transfected versus untransfected cells (0.0556±0.0018 vs.0.002±0.0002, p<0.0001, n=4 replicates), verifying in vitro its highspecificity for TREM-1. Blocking studies with unlabeled anti-TREM-1 mAbled to a significant reduction of [⁶⁴Cu]TREM-1 binding in transfectedcells (0.0556±0.0018 vs. 0.0167±0.0007, p<0.0001, n=4 replicates),further corroborating specificity of this PET imaging probe.

Example 3: In Vivo Imaging Studies

Methods: PET/CT imaging of middle cerebral artery occlusion (MCAo) micewas performed to investigate the feasibility of using [⁶⁴Cu]TREM-1 tovisualize neuroinflammation in vivo. We selected the MCAo model ofcerebral ischemia since the time-course of macrophage infiltration andmicroglial activation in the brain infarct is well documented, andbecause this model is commonly used to evaluate candidate microglial-PETtracers. B6 mice (n=3), MCAo (n=9), and sham (n=9) mice were injectedvia tail vein with 80-85 μCi of [⁶⁴Cu]TREM-1 in a saline solution (0.9%sodium chloride) and imaged using PET/CT at 3 h post-injection (FIG. 4).They were imaged again at 19 h post-injection, which was 1.5-2 daysafter surgery/stroke (FIG. 5).

Results: FIGS. 5, 6, and 7 show PET/CT images and quantitation of thebrain images and the respective uptake of TREM-1 in each brainhemisphere. PET/CT imaging results show high [⁶⁴Cu]TREM-1-PET signal inthe ischemic brain region of mice with established cerebral infarction(2.64±0.31% ID/g, n=9) compared to the corresponding contralateral(healthy) brain regions (1.79±0.14% ID/g) at early stages of cerebralischemia (i.e., 1.5-2 days post-stroke). There was negligible, if any,[⁶⁴Cu]TREM-1 PET signal in the brain of sham-operated mice (n=9). Toverify the penetration of our TREM-1-PET tracer into the brain, weperfused each mouse with saline after imaging, to remove possibleunbound intravascular [⁶⁴Cu]TREM-1, and then harvested brain tissue forex vivo gamma counting. These results revealed a 1.97-fold higheraccumulation of our TREM-1-PET tracer in the left (ischemic) hemispherecompared to the right side, and no difference between brain hemispheresof sham mice (MCAo=1.97±0.25, n=10; Sham=0.98±0.13, n=9). Since theseresults were comparable to our PET image quantitation, we believe[⁶⁴Cu]TREM-1 is able to sufficiently penetrate the brain of MCAO mice atthis time point. Taken together, these results indicate that[⁶⁴Cu]TREM-1 is a promising PET imaging probe that can detect regionscontaining neuroinflammation with high sensitivity and specificity.

Example 1-3 References

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Development and    validation companion diagnostic agent for antibody-drug conjugate    therapy to target the CA6. Radiology. 2015; 276(1):191-8. PMID:    25734548.-   34. Konishi S, Hamacher K, Vallabhajosula S, Kothari P, Bastidas D,    Bander N, Goldsmith S. Determination of immunoreactive fraction of    radiolabeled monoclonal antibodies: What is an appropriate method?    Cancer Biother Radiopharm. 2004; 19(6):706-15. PMID: 15665617.-   35. Zeng H, Ornatowska M, Joo M S, Sadikot R T. TREM-1 expression in    macrophages is regulated at transcriptional level by NFκB and PU.1.    Eur J Immunol. 2007; 37(8):2300-8. PMID: 17634956.-   36. Chen L C, Laskin J D, Gordon M K, Laskin D L. Regulation of TREM    expression in hepatic macrophages and endothelial cells during acute    endotoxemia. Exp Mol Pathol. 2008; 84(2):145-55. PMID: 18222421.-   37. McCullough L, Wu L, Haughey N, Liang X, Hand T, Wang Q, Breyer R    M, Andreasson K. Neuroprotective function of the PGE2 EP2 receptor    in cerebral ischemia. J Neurosci. 2004; 24(1):257-68. PMID:    14715958.-   38. Liang X, Lin L, Woodling N S, Wang Q, Anacker C, Pan T, Merchant    M, Andreasson K. Signaling via the prostaglandin E2 receptor EP4    exerts neuronal and vascular protection in a mouse model of cerebral    ischemia. J Clin Invest. 2011; 121(11):4362-71. PMID: 21965326.-   39. Li J, Liang X, Wang Q, Breyer R M, McCullough L, Andreasson K.    Misoprostol, an anti-ulcer agent and PGE2 receptor agonist, protects    against cerebral ischemia. Neurosci Lett. 2008; 438(2):210-5. PMID:    18472336.-   40. Liu C H, Huang S, Kim Y R, Rosen B R, Liu P K. Forebrain    ischemia-reperfusion simulating cardiac arrest in mice induces edema    and DNA fragmentation in the brain. Mol Imaging. 2007; 6(3):156-70.    PMID: 17532882.-   41. James M L, Fulton R R, Vercoullie J, Henderson D J. DPA-714, a    new translocator protein-specific ligand: Synthesis,    radiofluorination, and pharmacologic characterization. J Nucl Med.    2008; 49(5):814-22. PMID: 18413395.-   42. James M L, Belichenko N P, Nguyen T-V V., Andrews L A, Ding Z,    Liu H, Bodapati D, Arksey N, Shen B, Cheng Z, Wyss-Coray T, Gambhir    S S, Longo F M, Chin F T. PET imaging of translocator protein (18    kDa) in a mouse model of Alzheimer's disease using    N-(2,5-Dimethoxybenzyl)-2-18F-Fluoro-N-(2-Phenoxyphenyl)Acetamide. J    Nucl Med. 2015; 56(2):311-6. PMID: 25613536.

Example 4 Materials and Methods

DOTA Conjugation.

Conjugation of anti-mouse anti-TREM mAb (which can be purchased from R&DSystems) with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) was performed according to standard published procedures usingmetal-free buffers (1,2). Briefly, a solution of DOTA-NHS ester(Macrocyclics Inc.) in dimethyl sulfoxide (25 mmol/L; 9-12 μL) was addedto 1 mL of HEPES buffer (0.1 mol/L, pH 8.8) containing 500 μg ofTREM-1-mAb, and the reaction mixture was incubated at 4° C. overnight.The reaction was quenched with Tris (Sigma), excess DOTA-NHS was removedby Zeba Spin Desalting Columns (0.5 mL, 50K MWCO, ThermoFisherScientific), and the resulting solution was buffer-exchanged intoammonium acetate buffer (0.1 M, pH 5.5) for ⁶⁴Cu labeling.DOTA-conjugate solutions were concentrated by ultrafiltration (Vivaspin2 mL, Sartorius) to 2-5 mg/mL, snap frozen in liquid nitrogen, andstored at −80° C. prior to radiolabeling. The number of DOTA chelatorscoupled per TREM-1 was estimated to be 1.5-3.2, via matrix-assistedlaser desorption/ionization (MALDI) mass spectrometry.

Radiolabeling.

DOTA-TREM-1-mAb was radiolabeled with ⁶⁴Cu (t %=12.7 h) using standardmethods and metal-free buffers, with some modifications (1,2). In brief,DOTA-TREM-1-mAb (100 μg) in 50 μL of 0.25 mol/L ammonium acetate buffer(0.1 M, pH 5.5) was mixed with pH-balanced ⁶⁴CuCl₂ solution (44-74 MBq,pH 5.0-5.5, University of Wisconsin, Madison) at 37° C. with gentleshaking at 300 rpm. After a 60-minute incubation period, 0.1 M EDTA (0.5M, pH 8.0) was added to a final concentration of 0.01 M and incubated atroom temperature for 15 min to scavenge unchelated ⁶⁴CuCl₂ in thereaction mixture. Purification of ⁶⁴Cu-DOTA-TREM-1-mAb was achieved byG25 Sephadex size-exclusion purification (NAP-5 column). Radiochemicalpurity was determined by instant thin-layer chromatography withTEC-Control Chromatography strips (Biodex Medical Systems, Shirley,N.Y.), developed in saline, and size-exclusion liquid chromatographywith a Phenomenex SEC 3000 column (Torrance, Calif., USA) with sodiumphosophate buffer [0.1 mol/L, pH 6.8)] at a flow rate of 1.0 mL/min.⁶⁴Cu-labeled anti-TREM1 mAb (i.e., [⁶⁴Cu]TREM1-mAb) was obtained withhigh specific radioactivity (>75 GBq/μmol), radiochemical purity (>99%),and labeling efficiency (70-95%), and formulated in phosphate-bufferedsaline [0.1 mol/L NaCl, 0.05 mol/L sodium phosphate (pH 7.4)].

In Vitro Cell Uptake Studies with HEK293 Cells.

The uptake of [⁶⁴Cu]TREM1-mAb in HEK293 cells transfected with TREM1 wascompared to uptake in untransfected cells, in 24-well plates. One dayafter transfection, fresh, pre-warmed DMEM containing 0.925 MBq of[¹¹C]DASA-23 was added to individual wells (1 mL/well; 9.25±4.63 pmol).Cells were incubated with [⁶⁴Cu]TREM1-mAb at 37° C. and 5% CO₂ over a 60and 120 min time course. At the respective time points, plates wereplaced on ice, washed 3 times with phosphate-buffered saline (PBS), andlysed RIPA buffer (500 μL). 150 μL cell lysates were transferred tocounting tubes and decay-corrected radioactivity was determined on a γcounter (Cobra II Auto-Gamma counter; Packard Biosciences Co.). Theremaining lysate was frozen and used following radioactive decay forprotein determination using a bicinchoninic acid (BCA) 96-well plateassay (Thermo Fisher Scientific Inc.) In addition, 100 μL standards fromthe 0.925 MBq/mL solution added to cells were counted to quantitatepercentage radiotracer uptake.

In Vitro Cell Uptake Studies Using an Immortalized Murine MicroglialCell Line.

BV2 cells were incubated with either [⁶⁴Cu]TREM1-mAb (n=4 wells) or[⁶⁴Cu]-Isotype-control-mAb (n=4 wells) at 37° C. and 5% CO₂ for 120minutes. A sub-set of cells (n=4 separate wells for each tracer) werepre-treated with 100-fold unconjugated TREM1-mAb (compared to massamount associated with the tracer dose per well) for 30 min prior toadding radiotracer to serve as a blocking study to evaluate specificityof tracer binding. After incubating cells with each tracer for 120 min,plates were washed 3 times with phosphate-buffered saline (PBS), andlysed RIPA buffer (500 μL). 150 μL cell lysates were transferred tocounting tubes and decay-corrected radioactivity was determined on a γcounter (Cobra II Auto-Gamma counter; Packard Biosciences Co.). Inaddition, 50 μL standards from the radiotracer stock media solutionadded to cells were counted to quantitate percentage radiotracer uptake.After samples were counted, a bicinchoninic acid (BCA) 96-well plateassay (Thermo Fisher Scientific Inc.) was used to determine the amountof protein within each sample. The radioactive signal was laternormalized to the amount of protein within each sample.

LPS-Induced Sepsis in C57BL/6 Mice.

Lipopolysaccharide (LPS) from Escherichia coli lyophilized powder(Sigma) was dissolved in sterile saline immediately prior to injectingC57BL/6 female mice (8-12 weeks) intraperitoneally (i.p.) at aconcentration of 5 mg/kg. Vehicle mice received the same volumes ofsterile saline (according to their weight).

PET/CT Imaging of LPS and Vehicle Mice.

LPS and vehicle mice were injected with [⁶⁴Cu]TREM1-mAb (1.59±0.09 MBq)intravenously (i.v.). Mice were then imaged at 3 hours and 24 hours posti.v. injection. Mice were anesthetized using isoflurane gas (2.0-3.0%for induction and 1.5-2.5% for maintenance). A CT image was acquiredjust before each PET scan to provide an anatomic reference frame for therespective PET data. CT raw images were acquired at 80 kVp at 500 ρA,two bed position, half-scan 220° of rotation, and 120 projections perbed position with a cone beam micro-X-ray source (50 μm focal spot size)and a 2048×3072 pixel X-ray detector. On the basis of attenuationcorrection from the CT measurements, each 10-minute static PET scan wasacquired with default settings of coincidence, a timing window of 3.4ns, and an energy window of 350 to 650 keV. PET and CT Image files wereco-registered and analyzed with Inveon Research Workspace software (IRW,version 4.0; Siemens).

Biodistribution.

After the final PET scan, mice were deeply anesthetized with 2-2.5%isoflurane, and blood samples (100-200 μL) were collected via cardiacpuncture immediately prior to transcardial perfusion with 20 mL of PBS.Following perfusion, the heart, lungs, liver, spleen, kidney, smallintestine, bone (femur) and muscle were dissected from each mouse,placed in a tube for gamma counting, and weighed; satisfactoryperfusions were verified by visual inspection of brain tissue.Tissue-associated radioactivity in each dissected organ was assessed viaan automated gamma counter (Cobra II Auto-Gamma counter; PackardBiosciences Co.), normalized to tissue weight and to amount ofradioactivity administered to each mouse, and decay-corrected to time oftracer injection using diluted aliquots of the initial administered doseas standards.

Image Analysis.

Regions of interest (ROIs) were drawn around the spleen, liver, and bone(femur) using Inveon Research Workspace (IRW, version 4.0; Siemens).These ROIs were then normalized to the amount of radioactivityadministered to each mouse and decay-corrected to the time of scanning.

Results

After labeling DOTA-TREM-mAb with ⁶⁴Cu we assessed the in vitro bindingaffinity, specificity, and immunoreactivity of this new PET tracer usingstandard protocols in HEK293 cells+/−TREM transfection and in murinemicroglia+/−LPS treatment. In studies using HEK293 cells,[⁶⁴Cu]TREM1-mAb displayed 24-fold higher binding in transfected versusuntransfected cells (FIG. 1) (0.556±0.002 vs 0.002±0.0002, p<0.001, n=4replicates), confirming in vitro its high specificity for TREM1.Blocking studies with unlabeled anti-TREM1 mAb led to a significantreduction of [⁶⁴Cu]TREM1-mAb binding in transfected cells (0.556±0.002vs. 0.017±0.001, p<0.001, n=4 replicates), further corroborating thespecificity of our new PET tracer.

In vitro cell studies using BV2 cells, an immortalized murine microglialcell line, showed [⁶⁴Cu]TREM1-mAb binds specifically to TREM1 in thesecells, as demonstrated by the significantly higher uptake of this tracerin BV2 cells compared to those pre-treated for 30 min withunconjugated-TREM1-mAb (100-fold dose of PET tracer). [⁶⁴Cu]TREM1-mAbdisplayed 2.4 higher binding in BV2 cells compared to blocked BV2 cells(0.00045±2.99×10⁻⁵ vs. 0.00019±2.03×10⁻⁵, p<0.001, n=4 replicates). BV2cells incubated with [⁶⁴Cu]Isotype-Control PET tracer showedsignificantly lower uptake than BV2 cells incubated with [⁶⁴Cu]TREM1-mAb(0.000454±2.99×10⁻⁵ vs. 0.000253±0.0001, p<0.001, n=4 replicates). Theuptake of [⁶⁴Cu]isotype-control PET tracer in BV2 cells was comparableto [⁶⁴Cu]TREM1-mAb uptake in BV2 cells that had been pre-blocked,further confirming that the high uptake we observed with [⁶⁴Cu]TREM1-mAbin BV2 cells is due to specific binding to TREM1.

Furthermore, the uptake among [⁶⁴Cu]Isotype-Control PET tracer wascomparable to uptake in cells that had been pre-blocked, furtherconfirming that the high uptake we observed with [⁶⁴Cu]TREM1-mAb in BV2cells is due to specific binding to TREM1.

Following in vitro studies, we performed in vivo PET imaging and ex vivobiodistribution studies with [⁶⁴Cu]TREM1-mAb in a mouse model ofLPS-induced sepsis. These studies involved intravenous administration of[⁶⁴Cu]TREM1-mAb (90-100 μCi) immediately after intraperitoneal (i.p.)injection of either LPS (5 mg/kg) or vehicle. After 24 h of allowing thetracer to circulate and bind TREM1 in vivo, mice underwent a 10 minstatic PET/CT image and were then deeply anesthetized, perfused withsaline to remove possible unbound intravascular [⁶⁴Cu]TREM1-mAb, andorgans were harvested for gamma counting to determine percentageinjected dose per gram (% ID/g) of [⁶⁴Cu]TREM1-mAb bound in thesetissues.

In vivo PET imaging results (FIG. 2) demonstrated markedly higher levelsof [⁶⁴Cu]TREM1-mAb uptake in spleen, liver, and lungs compared tovehicle-treated mice. This pattern of TREM1 PET tracer uptake fits withthe known increase in splenic myeloid cells that occurs after LPSchallenge, and since lung and liver are among the first organs to beaffected/inflamed in sepsis. Interestingly, we observed lower TREM1 PETtracer uptake in bones of mice with LPS-induced sepsis compared tovehicle-treated mice, possibly highlighting the mobilization ofTREM1-positive myeloid cells from bone marrow to affected organs.

Quantitation of PET images (FIG. 3) acquired 24 h after mice receivedi.p. injection of LPS (5 mg/kg) or saline alone revealed significantlyhigher uptake of [⁶⁴Cu]TREM1-mAb in spleen and liver of LPS-treated(n=8) compared to vehicle-treated mice (n=5) and LPS mice that wereinstead injected with a radiolabeled isotype control PET tracer (n=3).LPS mice injected with radiolabeled isotype control mAb hadsignificantly lower uptake in spleen and bone marrow compared to LPSmice injected with [⁶⁴Cu]TREM1-mAb.

Ex vivo biodistribution results (FIG. 4) reveal significantly higheruptake of [⁶⁴Cu]TREM1-mAb in brain, lung, liver, and spleen of mice withLPS-induced sepsis, as per gamma counting of tissues 24 h after micereceived i.p. injection of LPS (5 mg/kg) or vehicle, n=3 per group.Importantly, we also observed 1.5-fold higher brain uptake of[⁶⁴Cu]TREM1-mAb in LPS-injected mice (0.19±0.02 vs. 0.13±0.01% ID/g,p<0.05, n=3).

Example 4 References

-   (1) Ilovich O., Natarajan A., Hori S., et al. Development and    Validation Companion Diagnostic Agent for Antibody-Drug Conjugate    Therapy to Target the CA6. 2015; 276:191-198. PMID: 25734548-   (2) Cooper M S., Ma M T., Sunassee K., et al. Comparison of    64cu-complexing bifunctional chelators for radioimmunoconjugation:    Labeling efficiency, specific activity, and in vitro/in vivo    stability. Bioconjug Chem. 2012; 23:1029-1039. PMID: 22471317

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. In an embodiment, “about 0” can refer to 0, 0.001,0.01, or 0.1. In an embodiment, the term “about” can include traditionalrounding according to significant figures of the numerical value. Inaddition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about‘y’”.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare set forth only for a clear understanding of the principles of thedisclosure. Many variations and modifications may be made to theabove-described embodiments of the disclosure without departingsubstantially from the spirit and principles of the disclosure. All suchmodifications and variations are intended to be included herein withinthe scope of this disclosure.

We claim:
 1. A method of imaging an inflammatory disease in a subjectcomprising: administering to a subject a labeled probe in a detectablyeffective amount, wherein the labeled probe includes an agent having anaffinity for TREM-1 and a radiolabel, imaging at least a portion of thesubject; and detecting the labeled probe, wherein the location of thelabeled probe corresponds to inflammation corresponding to theinflammatory disease.
 2. The method of claim 1, wherein the inflammatorydisease is selected from the group consisting of: Alzheimers disease,multiple sclerosis, epilepsy, traumatic brain injury, cancer, arthritis,inflammatory bowel disease, Huntington's disease, ALS, Parkinson'sdisease, an infectious disease, sepsis, pain, stroke, chronic fatiguesyndrome, depression, schizophrenia, and a CNS or peripheralinflammatory disease.
 3. The method of claim 1, wherein the inflammatorydisease is Alzheimers disease.
 4. The method of claim 1, wherein theinflammatory disease is sepsis.
 5. The method of claim 1, wherein theradiolabel is selected from the group comprising: ⁶⁴Cu, ¹²⁴I,^(76/77)Br, ⁸⁶Y, ⁸⁹Zr, ⁶⁸Ga, ¹⁸F, 11C, ¹²⁵I, ¹²⁴I, ¹³¹I, ¹²³I, ³²Cl,³³Cl, ³⁴Cl, ⁶⁸Ga, ⁷⁴Br, ⁷⁶Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y,⁸⁶Y, ¹⁷⁷Lu, and ¹⁵³Sm.
 6. The method of claim 1, wherein the radiolabelis conjugated to the agonist antibody of TREM-1 using a chelatorselected from the group consisting of:(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); NOTA(1,4,7-Triazacyclononane-1,4,7-triacetic acid); EDTA(Ethylenediaminetetraacetic acid); Df (Desferrioxamine); DTPA(Diethylenetriaminepentaacetic acid; and TETA (Triethylenetetramine). 7.The method of claim 1, wherein the agent having an affinity for TREM-1is selected from a TREM-1 antibody, a TREM-1 peptide, and a smallmolecule associated with TREM-1.
 8. The method of claim 7, wherein theTREM-1 antibody is selected from a TREM-1 agonist antibody or anantagonist TREM-1 antibody.
 9. The method of claim 1, wherein theradiolabel is selected from the group comprising: ⁶⁴Cu, ¹²⁴I,^(76/77)Br, ⁸⁶Y, ⁸⁹Zr, ⁶⁸Ga, ¹⁸F, ¹¹C, ¹²⁵I, ¹²⁴I, ¹³¹I, ¹²³I, ³²Cl,³³Cl, ³⁴Cl, ⁶⁸Ga, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y,⁸⁶Y, ¹⁷⁷Lu, and ¹⁵³Sm.
 10. (canceled)
 11. A method of monitoring theprogress of an inflammatory disease in a subject, the method comprising:administering to a subject a labeled probe, wherein the labeled probeincludes an agent having an affinity for TREM-1 and a radiolabel,imaging at least a portion of the subject; and detecting the labeledprobe, wherein the location of the labeled probe corresponds toinflammation corresponding to the inflammatory disease, wherein thedimensions of the location are monitored over time.
 12. The method ofclaim 11, wherein the level of uptake of the labeled probe in a tissuecorresponds to the level inflammation in the tissue.
 13. The method ofclaim 11, wherein the detection of the labeled probe is performed invitro using Positron Emission Tomography (PET), Computerized Tomography(CT), or a combination thereof.
 14. (canceled)
 15. The pharmaceuticalcomposition of claim 18, wherein the radiolabel is selected from thegroup comprising: ⁶⁴Cu, ¹²⁴I, ^(76/77)Br, ⁸⁶Y, ⁸⁹Zr, ⁶⁸Ga, ¹⁸F, ¹¹C,¹²⁵I, ¹²⁴I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁶⁸Ga, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,⁷⁸Br, ⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ⁸⁶Y, ¹⁷⁷Lu, and ¹⁵³Sm.
 16. Thepharmaceutical composition of claim 18, wherein the radiolabel isconjugated to the agent having an affinity for TREM-1 using a chelatorselected from the group consisting of:(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); NOTA(1,4,7-Triazacyclononane-1,4,7-triacetic acid); EDTA(Ethylenediaminetetraacetic acid); Df (Desferrioxamine); DTPA(Diethylenetriaminepentaacetic acid; and TETA (Triethylenetetramine).17. The pharmaceutical composition of claim 18, wherein the radiolabelis ⁶⁴Cu and is conjugated to the agent having an affinity for TREM-1using DOTA.
 18. A pharmaceutical composition, comprising: apharmaceutical carrier and an effective dose of a labeled probe, whereinthe labeled probe includes an agent having an affinity for TREM-1 and aradiolabel.
 19. The pharmaceutical composition of claim 18, wherein theradiolabel is conjugated to the agonist antibody of TREM-1 using achelator selected from the group consisting of:(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); NOTA(1,4,7-Triazacyclononane-1,4,7-triacetic acid); EDTA(Ethylenediaminetetraacetic acid); Df (Desferrioxamine); DTPA(Diethylenetriaminepentaacetic acid; and TETA (Triethylenetetramine).